Study Data
| Project uploaded by: | Yashwant |
| Project ID: | IMP_100044 |
| Title: | (p)ppGpp and DksA play crucial role in reducing the efficacy of ꞵ-lactam antibiotics by modulating bacterial membrane permeability |
| Project Description: | The key signaling molecules in the bacterial stress sensing pathway, the alarmone (p)ppGpp and transcription factor DksA, help in survival during nutritional deprivation and exposure to xenobiotics by modulating cellular metabolic pathways. In Vibrio cholerae, (p)ppGpp metabolism is solely linked with the functions of three proteins: RelA, SpoT, and RelV. At threshold or elevated concentrations of (p)ppGpp, the level of cellular metabolites and proteins in the presence and absence of DksA in V. cholerae and other bacteria has not yet been comprehensively studied. We engineered the genome of V. cholerae to develop DksA null mutants in the presence and absence of (p)ppGpp biosynthetic enzymes. We observed a higher sensitivity of the (p)ppGpp0ΔdksA V. cholerae mutant to different ꞵ-lactam antibiotics compared to the wild-type (WT) strain. Our whole-cell metabolomic and proteome analysis revealed that the cell membrane and peptidoglycan biosynthesis pathways are significantly altered in the (p)ppGpp0, ΔdksA, and (p)ppGpp0ΔdksA V. cholerae strains. Further, the mutant strains displayed enhanced inner and outer membrane permeability in comparison to the WT strains. These results directly correlate with the tolerance and survival of V. cholerae to ꞵ-lactam antibiotics. These findings may help in the development of adjuvants for ꞵ-lactam antibiotics by inhibiting the functions of stringent response modulators. |
| Research Area: | Biological Sciences |
| Funding Source: | Translational Research Program (TRP) (No. BT/PR30159/MED/15/188/2018) of Department of Biotechnology (DBT), Govt. of India. |
| Project Contributors: | Yashwant Kumar |
| Sr.No | Sample ID | Sample Name | Organism | Source | Sample Preparation Protocol | Sample Type | Experimental Condition | Time of treatment | Variant/Variety | Gender | Age | Replicates | Storage Conditions | Extraction Protocol | Number of files per sample |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 71 | IMSM_102121 | N16_5 | Vibrio cholerae | 666 | Bacteria | The cells were pelleted down again by centrifugation (10,000 rpm at 4°C for 10 min), washed with 0.9% normal saline and stored at -80°C. To extract the intracellular metabolites cold 100% methanol was added (Sigma Aldrich; Cat no. 34860) followed by vortexing and bath sonication for 10 min (Bransonic® Ultrasonic M Cleaning Bath 1510). The cell debris was pelleted down by centrifugation (10,000 rpm at 4°C for 10 min) and supernatant was collected in two separate microcentrifuge tubes (120 µL each tube), vacuum dried (Thermo Scientific™ Savant™ SPD1010) and stored at -80°C. For the analysis of metabolites, the dried supernatant was dissolved in 60 µL of 15% methanol or 50% acetonitrile (Cat no. 271004) followed by vortexing for 5 min and centrifuged (10,000 rpm for 10 min). The supernatant was collected in a separate sample vial (Supelco™ Analytical). | wild type | Untreated | overnight at 37°C | NA | NA | NA | NA | –80ºC |
The cells were pelleted down again by centrifugation (10,000 rpm at 4°C for 10 min), washed with 0.9% normal saline and stored at -80°C. To extract the intracellular metabolites cold 100% methanol was added (Sigma Aldrich; Cat no. 34860) followed by vortexing and bath sonication for 10 min (Bransonic® Ultrasonic M Cleaning Bath 1510). The cell debris was pelleted down by centrifugation (10,000 rpm at 4°C for 10 min) and supernatant was collected in two separate microcentrifuge tubes (120 µL each tube), vacuum dried (Thermo Scientific™ Savant™ SPD1010) and stored at -80°C. For the analysis of metabolites, the dried supernatant was dissolved in 60 µL of 15% methanol or 50% acetonitrile (Cat no. 271004) followed by vortexing for 5 min and centrifuged (10,000 rpm for 10 min). The supernatant was collected in a separate sample vial (Supelco™ Analytical). |
4 |
| 72 | IMSM_102122 | N16_6 | Vibrio cholerae | 666 | Bacteria | The cells were pelleted down again by centrifugation (10,000 rpm at 4°C for 10 min), washed with 0.9% normal saline and stored at -80°C. To extract the intracellular metabolites cold 100% methanol was added (Sigma Aldrich; Cat no. 34860) followed by vortexing and bath sonication for 10 min (Bransonic® Ultrasonic M Cleaning Bath 1510). The cell debris was pelleted down by centrifugation (10,000 rpm at 4°C for 10 min) and supernatant was collected in two separate microcentrifuge tubes (120 µL each tube), vacuum dried (Thermo Scientific™ Savant™ SPD1010) and stored at -80°C. For the analysis of metabolites, the dried supernatant was dissolved in 60 µL of 15% methanol or 50% acetonitrile (Cat no. 271004) followed by vortexing for 5 min and centrifuged (10,000 rpm for 10 min). The supernatant was collected in a separate sample vial (Supelco™ Analytical). | wild type | Untreated | overnight at 37°C | NA | NA | NA | NA | –80ºC |
The cells were pelleted down again by centrifugation (10,000 rpm at 4°C for 10 min), washed with 0.9% normal saline and stored at -80°C. To extract the intracellular metabolites cold 100% methanol was added (Sigma Aldrich; Cat no. 34860) followed by vortexing and bath sonication for 10 min (Bransonic® Ultrasonic M Cleaning Bath 1510). The cell debris was pelleted down by centrifugation (10,000 rpm at 4°C for 10 min) and supernatant was collected in two separate microcentrifuge tubes (120 µL each tube), vacuum dried (Thermo Scientific™ Savant™ SPD1010) and stored at -80°C. For the analysis of metabolites, the dried supernatant was dissolved in 60 µL of 15% methanol or 50% acetonitrile (Cat no. 271004) followed by vortexing for 5 min and centrifuged (10,000 rpm for 10 min). The supernatant was collected in a separate sample vial (Supelco™ Analytical). |
4 |
| Sr.No | MS Exp ID | Sample Name/ID | Mass Spectrometer Type | MS Instrument Name | MS Instrument type | MS Ionization Method | Ion Mode/Scan Polarity | Data Transformation (Software/s Used) |
|---|---|---|---|---|---|---|---|---|
| 1 | IME_101273 | JV7_1 / IMSM_102057 | LCMS (Liquid Chromatography- Mass Spectrometry) | Thermo Fusion Orbitrap | Orbitrap | Electrospray Ionization - ESI | Negative | NA |
| 2 | IME_101274 | JV7_2 / IMSM_102058 | LCMS (Liquid Chromatography- Mass Spectrometry) | Thermo Fusion Orbitrap | Orbitrap | Electrospray Ionization - ESI | Negative | NA |
| 3 | IME_101275 | JV7_3 / IMSM_102059 | LCMS (Liquid Chromatography- Mass Spectrometry) | Thermo Fusion Orbitrap | Orbitrap | Electrospray Ionization - ESI | Negative | NA |
| 4 | IME_101276 | JV7_4 / IMSM_102060 | LCMS (Liquid Chromatography- Mass Spectrometry) | Thermo Fusion Orbitrap | Orbitrap | Electrospray Ionization - ESI | Negative | NA |
| 5 | IME_101277 | JV7_5 / IMSM_102061 | LCMS (Liquid Chromatography- Mass Spectrometry) | Thermo Fusion Orbitrap | Orbitrap | Electrospray Ionization - ESI | Negative | NA |
| 6 | IME_101278 | JV7_6 / IMSM_102062 | LCMS (Liquid Chromatography- Mass Spectrometry) | Thermo Fusion Orbitrap | Orbitrap | Electrospray Ionization - ESI | Negative | NA |
| 7 | IME_101279 | JV8_1 / IMSM_102063 | LCMS (Liquid Chromatography- Mass Spectrometry) | Thermo Fusion Orbitrap | Orbitrap | Electrospray Ionization - ESI | Negative | NA |
| 8 | IME_101280 | JV8_2 / IMSM_102064 | LCMS (Liquid Chromatography- Mass Spectrometry) | Thermo Fusion Orbitrap | Orbitrap | Electrospray Ionization - ESI | Negative | NA |
| 9 | IME_101281 | JV8_3 / IMSM_102065 | LCMS (Liquid Chromatography- Mass Spectrometry) | Thermo Fusion Orbitrap | Orbitrap | Electrospray Ionization - ESI | Negative | NA |
| 10 | IME_101282 | JV8_4 / IMSM_102066 | LCMS (Liquid Chromatography- Mass Spectrometry) | Thermo Fusion Orbitrap | Orbitrap | Electrospray Ionization - ESI | Negative | NA |
| Sr.No | First name | Last name | Organization | Designation | |
|---|---|---|---|---|---|
| 1 | Yashwant | Kumar | y.kumar@thsti.res.in | Translational Health Science And Technology Institute (THSTI) | scientist |
| Sr.No | ftprun ID | MS Exp ID | MS Data Files |
|---|---|---|---|
| 21 | IMR_101897 | IME_101293 | HILIC_NEG_MC3_3.mzXML |
| 22 | IMR_101898 | IME_101294 | HILIC_NEG_MC3_4.mzXML |
| 23 | IMR_101899 | IME_101295 | HILIC_NEG_MC3_5.mzXML |
| 24 | IMR_101900 | IME_101296 | HILIC_NEG_MC3_6.mzXML |
| 25 | IMR_101901 | IME_101297 | HILIC_NEG_MC4_1.mzXML |
| 26 | IMR_101902 | IME_101298 | HILIC_NEG_MC4_2.mzXML |
| 27 | IMR_101903 | IME_101299 | HILIC_NEG_MC4_3.mzXML |
| 28 | IMR_101904 | IME_101300 | HILIC_NEG_MC4_4.mzXML |
| 29 | IMR_101905 | IME_101301 | HILIC_NEG_MC4_5.mzXML |
| 30 | IMR_101906 | IME_101302 | HILIC_NEG_MC4_6.mzXML |